RU2803848C1 - Method for chromatographic purification of exosomes and their separation from influenza a virus virions based on hydrophobic reaction with a sorbent - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: method for chromatographic purification of exosomes and their separation from influenza A virus virions based on hydrophobic interaction with a sorbent based on cross-linked polyacrylamide granules is described. A concentrated sample is obtained from the cellular environment containing exosomes and viral particles. Sorption buffer is added to the sample. Chromatographic separation is carried out using a column packed with Sephacryl S-500HR sorbent. Then the exosomes are eluted. A buffer including 500 mM sodium chloride and 10 mM sodium phosphate is used, and elution of exosomes is carried out with deionized water.

EFFECT: providing the possibility of isolating exosomes from the cellular environment and separating them from influenza virus virions by a chromatographic method due to the hydrophobic interaction of exosomes with a sorbent based on cross-linked polyacrylamide granules.

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Description

Field of technology to which the invention relates

The invention relates to the field of biotechnology, namely to the isolation of extracellular vesicles (exosomes) secreted into the culture medium by cells infected with the influenza A virus by chromatography using a sorbent based on cross-linked polyacrylamide granules.

State of the art

Currently, exosomes are being actively studied and used in the field of molecular biology, cytology and biotechnology. Exosomes are nanosized (30-150 nm) membrane vesicles that are secreted by almost all types of cells and, as a result, are present in almost all biological fluids of the body, for example, the presence of exosomes has been shown in urine [1], saliva [2], and blood [ 3], breast milk [4], amniotic [5] and bronchoalveolar [6] fluids. It is known that these nanosized particles contain various types of biomolecules, including proteins, lipids, carbohydrates, as well as DNA and RNA [7].

A distinctive feature of exosomes is the ability to transfer functional proteins and nucleic acids between cells, as well as overcome various biological barriers, which determines one of the key functions of exosomes - participation in intercellular communication. Moreover, the composition of exosomes (the set and concentration of proteins, RNA and other biological molecules) can vary depending on the physiological or pathological processes occurring inside cells [8]. Thus, studying the composition of exosomes can be used in the future to develop diagnostic markers for various pathological conditions.

Exosomes play an important role in the life cycle of various viruses. In particular, exosomes help some evade the immune response, facilitating the spread of viral infection. The role of exosomes in the life cycles of viruses such as herpes simplex virus [9], human immunodeficiency [10], Epstein-Barr [11], hepatitis A and C [12,13] has been demonstrated. However, the literature devoted to the study of the composition and role of exosomes in the life cycle of the influenza virus is very limited, which is apparently due to the lack of a protocol for isolating exosomes not contaminated with viral particles from cells infected with the influenza virus. This is a consequence of the similarity of the physicochemical properties of exosomes and influenza virus virions (such as buoyant density, size, the presence of host proteins in the particles [14,15]), as well as the duration of one cycle of influenza virus replication in cells, which on average is 8 hours, whereas any exosome isolation protocols require their accumulation in the cellular environment for at least 24 hours [16].

Thus, due to the significant potential of exosomes in the field of modern diagnostics and therapy, as well as to improve understanding of their role in the body, including influenza infection, it is necessary to develop a method for isolating pure exosomes that do not contain viral particles.

There is a known method for isolating exosomes from the extracellular environment using ultracentrifugation of the cellular environment [16,17], which consists of two stages: 1) centrifugation of the environment at low speeds (about 3000g to clean the environment from cells and their debris, and then about 10000g to remove extracellular large vesicles), while the supernatant is used for subsequent steps, and the sediment is discarded; 2) ultracentrifugation of the medium at a speed of 100,000g to concentrate exosomes. However, this method cannot be used to separate exosomes and influenza viruses and obtain a pure fraction of exosomes, since objects of the same size are isolated together by this method, and it is known that the size of exosomes is 30-150 nm [18], influenza viruses - about 100 nm .

There is also a known method for isolating exosomes using a method due to the specific interaction of antibodies with the surface proteins of exosomes, that is, using immunoaffinity chromatography [19]. Magnetic or polymer particles functionalized with specific antibodies, most often to tetraspanins (CD9, CD63 and CD81), are used as a stationary phase. This method turns out to be expensive due to the use of antibodies, and therefore its use is not justified for isolating exosomes from large volumes of biological material. At the same time, this technique is also not applicable for the separation of exosomes and viruses: the biogenesis of exosomes and influenza virus is similar, and therefore the compositions of cellular proteins on the surface of exosomes and influenza virus virions are also similar, which does not allow them to be separated due to differences in the set surface proteins.

There is also a known method for isolating exosomes using gel filtration chromatography. In this technique, the original biological fluid acts as the mobile phase, and the porous gel filtration polymer acts as the stationary phase. The properties of the stationary phase (for example, pore diameter) determine the rate of movement of particles of different sizes in the column: large particles leave the column earlier, small ones later, due to which separation by size is achieved, that is, it is possible to separate a fraction corresponding to exosomes in size. Cross-linked dextrans (Sephadex), agarose (Sepharose), polyacrylamide (Biogel P) or allyldextran (Sephacryl) are used as the stationary phase [20]. A method for isolating exosomes from cells by gel filtration using Sephacryl S500HR (Sigma) sorbent is described [21], where the first stage involves concentrating the sample (culture medium) by tangential filtration using the Akta flux system (GE Healthcare) and an MWCO filter (molecular weight cut-off) with a pore size of 750 kDa, and at the second stage, gel filtration is carried out using an Akta pure chromatograph (GE Healthcare) and a column containing Sephacryl S500HR. During tangential filtration, the culture medium was transferred to phosphate-buffered saline, pH 7.4, and then acted as a mobile phase for chromatography on a column pre-equilibrated with phosphate-buffered saline. Elution was also carried out with phosphate-buffered saline, after which the fractions corresponding to the first peak were collected and pooled. These combined fractions contained particles corresponding in size and marker proteins to exosomes. This method of isolating exosomes from the cellular environment by gel filtration using Sephacryl S500HR sorbent (Sigma) was accepted as the closest analogue.

There is also a known method for isolating variants of the recombinant influenza virus nucleoprotein using affinity chromatography on heparin-Sepharose followed by gel chromatography using Sephacryl S300 [22], characterized in that Sephacryl S300 was used as a classical sorbent for fractionation by hydrodynamic radius, and not for fractionation by due to hydrophobic (or some other) interaction, which does not allow the separation of exosomes and influenza virus particles.

From the above analogues, it is known to isolate exosomes from a culture medium, and it is also known to use the gel filtration method, including the use of Sephacryl sorbent. At the same time, to date there is no known method for isolating exosomes produced by cells infected with influenza A virus from a culture medium that also contains influenza virus, that is, there is no known method for separating exosomes and virions of influenza A viruses.

Thus, the technical problem is the need to develop a technique that makes it possible to isolate exosomes (not contaminated with viral particles) from the cellular environment from cells infected with the influenza virus, which, in addition to exosomes, also contains influenza virus virions.

Disclosure of the invention

The technical result consists in providing the possibility of isolating exosomes from the cellular environment and separating them from influenza virus virions by a chromatographic method due to the hydrophobic interaction of exosomes with a sorbent based on cross-linked polyacrylamide granules.

The technical result is achieved by the fact that in the method of chromatographic purification of exosomes and their separation from influenza A virus virions based on hydrophobic interaction with a sorbent based on cross-linked polyacrylamide granules, during which a concentrated sample is obtained from the cellular environment containing exosomes and viral particles and added to the sample buffer for sorption, filtration is carried out using a chromatographic column packed with Sephacryl S-500HR sorbent, after which exosomes are eluted; according to the invention, a buffer containing 500 mM sodium chloride and 10 mM sodium phosphate is used, and exosomes are eluted with deionized water.

Brief description of drawings

Fig. 1. Results of electron microscopy of a sample obtained after isolation from the cellular environment of exosomes secreted by (A) control cells or (B) cells infected with influenza A virus. Arrows in (A) indicate exosomes, arrows in (B) indicate exosomes and influenza A viruses. “E” - exosomes, “VG” - influenza virus.

Fig. 2. Typical chromatogram of a sample containing exosomes. Red line - conductivity mS/cm; The violet line is the optical density at a wavelength of 280 nm. Arrows mark the beginning of elution with water and the beginning of exosome release. The yellow and blue rectangles indicate the fractions, united under the names “C” (salt) and “B” (water).

Fig. 3. Results of the analysis of fractions obtained after chromatography with the Sephacryl sorbent of pure exosomes, using different saline buffers (Buffer 1, Buffer 2 and Buffer 3). (A) Dot blot with exosome marker antibodies (CD9 and MFG-E8). Red arrows indicate where the sample was applied. C - fractions after washing with saline buffer, B - elution with water, PC - positive control, E - exosomes, AT - antibodies. (B) Transmission electron microscopy of the aqueous fraction obtained after sorption of exosomes in Buffer 3. White arrows indicate exosomes; the length of the scale bar is 200 nm.

Fig. 4. Results of dot blot analysis of fractions obtained after chromatography with Sephacryl sorbent of pure viruses using different saline buffers (Buffer 1 and Buffer 3), with antibodies to the nucleoprotein (NP) of influenza A virus (IAV). Red arrows indicate where the sample was applied. C - fractions after washing with saline buffer, B - elution with water, PC - positive control, AT - antibodies.

Fig. 5. Results of analysis of fractions obtained after chromatography with the Sephacryl sorbent of a mixture of exosomes and virus using Buffer 3. (A) Dot blot with marker antibodies of exosomes (CD9) and the surface protein hemagglutinin (HA) of influenza A virus (IAV). Red arrows indicate where the sample was applied. C - fractions after washing with saline buffer, B - elution with water, PC - positive control, E - exosomes, AT - antibodies. (B) Transmission electron microscopy of (top) the aqueous fraction obtained after sorption of exosomes in Buffer 3 and (bottom) the salt fraction. White arrows indicate exosomes (top) and influenza A viruses (bottom); the length of the scale bar is 200 nm.

Carrying out the invention

A technique has been proposed that makes it possible to separate exosomes and influenza virus particles when they are isolated from the cellular environment from cells infected with the influenza virus, which consists of the following steps:

1) Obtaining a concentrated sample from the cellular environment containing exosomes and viral particles.

2) Adding a buffer with high ionic strength (sorption buffer) to the sample.

3) Balancing the chromatographic column packed with Sephacryl S-500HR sorbent with sorption buffer.

4) Applying the sample to the column and washing with sorption buffer.

5) Elution of exosomes with deionized water.

The first stage - obtaining concentrated samples of exosomes secreted by influenza virus-infected cells - is carried out by any standard method and is not the subject of this invention. Cellular medium, serum or blood plasma, urine, bronchoalveolar or other biological fluid containing exosomes and influenza virus particles can be used as starting material. Concentration of exosomes can be carried out, for example, by sequential centrifugation of the cell medium to remove cell debris and microvesicles, and then concentration of exosomes and viruses by ultracentrifugation, as well as by tangential filtration, or other methods.

At the second stage, a buffer with high ionic strength (sorption buffer) is added to the sample containing exosomes contaminated with influenza virus virions, which later during chromatography will facilitate the binding of exosomes to the Sephacryl sorbent according to the hydrophobic principle and thereby ensure their retention on the sorbent. Influenza virus virions, in turn, are not able to bind to the sorbent according to the hydrophobic principle, so their retention will not occur. As such a sorption buffer, for example, a solution of 500 mM sodium chloride, 10 mM sodium phosphate, pH 7.4 (final concentration in the sample) can be used. Thus, the use of a buffer with high ionic strength for the mobile phase in the chromatography process distinguishes this method from the known method of isolating exosomes using gel filtration chromatography, and also determines not only the size separation in the chromatography process (as in the case of using a standard gel filtration buffer). filtration, for example, phosphate-buffered saline or the original biological fluid as a mobile phase), but also according to the principle of hydrophobicity, which determines the further separation of exosomes and influenza virus particles.

In the third stage, the chromatographic column, packed with a sorbent based on cross-linked polyacrylamide granules, for example, Sephacryl S500HR or S1000HR, preferably S500HR, is equilibrated with the sorption buffer used in the previous stage.

At the fourth stage, the sample in the sorption buffer is applied to the column and then washed with the sorption buffer. At this stage, exosomes bind to the Sephacryl sorbent due to hydrophobic interactions and are retained. In this case, influenza virus virions leave the column in a free volume (sample A).

At the fifth and final stage, the column with sorbed exosomes is washed with deionized water, as a result of which the exosomes elute and leave the column in the aqueous phase (sample B).

Next, the method for separating exosomes and influenza virus particles, based on the hydrophobic interaction of exosomes and the Sephacryl sorbent, will be described in the form of examples that are intended to illustrate the invention and in no way limit it.

The claimed invention is illustrated by examples.

Example 1. Obtaining a concentrated sample containing exosomes and influenza virus virions.

The A549 cell line (ATCC, CCl-185, human lung carcinoma cells) was used to accumulate exosomes. The cell line was maintained on a culture medium (DMEM Nutrient Medium, Biolot, Russia) with 10% bovine serum (FBS, Biowest, USA) and with the addition of an antibiotic/antimycotic. Cells were maintained in T300 flasks (Nunc™ 300 cm ² Cell Culture Treated Flasks, Thermo Fisher, USA). When 90% of the cell monolayer was reached, cells were infected with influenza virus A/Puerto Rico/8/1934 (H1N1) at an infectious dose of 1 moi (one virus per cell). The cell monolayer density for infection was 1.3*10 ⁵ cells/cm ² . For one T300 vial, one infectious dose was 3.9 * 10 ⁷ TUD 50. Afterwards, the cells were washed from the virus with phosphate-buffered saline, and medium without bovine serum was added. After incubation for 24 hours at +37°C in an atmosphere of 5% CO _2, the culture medium was collected and used for exosome isolation.

To obtain a control sample (exosomes that do not contain viral particles), at the stage of reaching 90% monolayer, A549 cells were not infected, but were transferred to a medium that did not contain fetal bovine serum, and then incubated similarly for 24 hours.

To concentrate exosomes, the culture medium was centrifuged at a speed of 3000g for 30 minutes at a temperature of +4°C in a Centrifuge 5810 R centrifuge (Eppendorf), and the sediment was removed. The collected supernatant was centrifuged again at 10,000 g for 30 minutes at +4°C in an Optima XPN centrifuge (Beckman Coulter), and the sediment was removed. The collected supernatant was centrifuged at 110,000g for 2 hours at +4°C in an Optima XPN centrifuge (Beckman Coulter). The resulting pellet containing exosomes (control sample, Fig. 1A) or a mixture of exosomes and influenza virus virions (Fig. 1B) was resuspended in phosphate-buffered saline.

Example 2. Chromatography with Sephacryl sorbent of pure exosomes (control sample) using buffers with different ionic strengths

To study the binding of exosomes to the Sephacryl sorbent according to the hydrophobic principle, three sorption buffers with different ionic strengths were compared: Buffer 1) 100 mM sodium chloride, 10 mM sodium phosphate, pH 7.4; Buffer 2) 250 mM sodium chloride, 10 mM sodium phosphate, pH 7.4; Buffer 3) 500 mM sodium chloride, 10 mM sodium phosphate, pH 7.4. We used the AKTA pure 25 chromatographic system (GE Healthcare) at a temperature of +5°C, a 5 ml chromatographic column (Biotoolomics) packed with Sephacryl S500HR sorbent. The system was equilibrated using 10 mL of sorption buffer at a flow rate of 1 mL/min. The test drug in a volume of 1 ml was introduced into the column through an injection loop, after which it was washed with 15 ml of sorption buffer, and then eluted with 2 ml of deionized water (with a flow rate of 0.25 ml/min). During washing and elution, 0.5 mL fractions were collected. A typical chromatogram is shown in Fig. 2. For further analysis, the fractions were combined into salt (“C”) and water (“B”). The resulting fractions were analyzed by transmission electron microscopy (to confirm the shape and size of the obtained particles) and dot blotting (to confirm the presence of marker proteins).

Dot blotting was performed with antibodies to exosome marker proteins (CD9, MFG-E8). From the results of dot blotting (Fig. 3A), it is clear that with increasing ionic strength of the buffer, the number of exosomes adsorbed on the column increases, since the points corresponding to the aqueous fractions (“B”) have greater intensity than the points corresponding to the salt fractions ( "WITH"). The greatest difference is observed for Buffer 3, which contains the highest concentration of sodium chloride. Using transmission electron microscopy, it was shown that the aqueous fraction obtained after sorption of exosomes in Buffer 3 contains exosomes (Fig. 3B), since cup-shaped particles with a size of about 80-100 nm are observed. Thus, it was shown that exosomes are present predominantly in aqueous fractions, and with an increase in the ionic strength of the solution, an increase in the concentration of exosomes in the aqueous fraction is observed; the maximum effect is achieved when using Buffer 3.

Example 3. Chromatography with Sephacryl influenza virus sorbent using buffers with different ionic strengths

Chromatography with a sample containing only the influenza A/Puerto Rico/8/1934 (H1N1) virus was carried out similarly to Example 2 using Buffer 1 and Buffer 3. Collection and pooling of fractions was also carried out in a similar manner. To detect the virus, dot blotting was carried out with the structural protein of the influenza virus - nucleoprotein (Fig. 4). It was shown that the nucleoprotein is present in the salt fraction when using Buffer 3, that is, it does not bind to Sephacril in a buffer with high ionic strength, unlike exosomes.

Example 4. Chromatography with Sephacryl sorbent of a mixture of exosomes and influenza virus using a buffer with high ionic strength

A mixture of exosomes and influenza virus was obtained as described in Example 1. Chromatography, collection and combination of fractions were carried out similarly to Example 2 using Buffer 3. To detect marker proteins of exosomes by dot blotting, antibodies to CD9 were used, and for the detection of influenza A virus, antibodies to the surface protein of the influenza virus, hemagglutinin. Based on the results of dot blotting with antibodies to hemagglutinin, it can be concluded that the influenza virus is present in the salt fraction (“C”) and absent in the water fraction (“B”), while the exosome marker protein CD9 (Fig. 5A) is detected in both the aqueous fraction , and in saline, which indicates the presence of exosomes in the aqueous fraction, as well as the presence of the CD9 protein in the structure of the influenza virus virion, which was previously described in the literature [15]. The resulting fractions were also analyzed by transmission electron microscopy to confirm the shape and size of the resulting particles (Figure 5B). The presence of exosomes in the aqueous fraction and influenza viruses in the saline fraction was shown. Thus, the use of a buffer with high ionic strength (containing 500 mM sodium chloride) allows the separation of exosomes and viral particles due to the interaction of exosomes with the Sephacryl sorbent according to the hydrophobic principle.

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Claims (1)

A method for chromatographic purification of exosomes and their separation from influenza A virus virions based on hydrophobic interaction with a sorbent based on cross-linked polyacrylamide granules, during which a concentrated sample is obtained from the cellular environment containing exosomes and viral particles, a sorption buffer is added to the sample, and chromatographic separation is carried out using a column packed with Sephacryl S-500HR sorbent, after which exosomes are eluted, characterized in that a buffer containing 500 mM sodium chloride and 10 mM sodium phosphate is used, and exosomes are eluted with deionized water.